The secretion of stored granular components, known as the release reaction, is a central event is platelet function. The reaction occurs in response to a variety of stimuli and leads, under appropriate conditions, to the formation of platelet aggregates. Consequently, secretion by platelets contributes in a major way to hemostatis. Platelets, like numerous other secretory cells, display a striking shift in a phosphoinositide (PI) metabolism when challenged with appropriate agonists, such as thrombin. The role played by PI in the secretory process has yet to be elucidated. However, perturbations in PI turnover are so consistently associated with stimulus-secretion that closer study of PI metabolism in platelets is warranted. Defects in PI metabolism may underlie certain abnormalities in human hemostasis and thrombosis. To dissect the various aspects of PI metabolism we propose to examine the factors regulating the synthesis and turnover of platelet PI, particularly those factors which 1) control a PI-specific phospholipase C (PI-PLC), whose existence has been reported by this laboratory and 2) regulate branch-point enzymes which in turn control PI synthesis. With regard to the first objective, we will purify PI-PLC further and examine the effects of calmodulin, Ca 2 ion, thrombin, and cAMP upon the activity of this enzyme. The fate of diglyceride, which we have observed to be generated rapidly from PI in response to secretory stimuli, will be investigated as well. In related studies, we intend to determine, through the use of phospholipase A (PLA) inhibitors, whether PI is hydrolyzed as well by PLA, and the extent to which PI-PLC or PLA contribute to prostaglandin production. We will also analyze the amount of PI at the exterior of the platelet membrane, the extent of synthesis of myoinositol by human platelets, and whether an association exists between PI and a platelet membrane protein component. In conjunction with this work, we plan to examine the phosphoinositide metabolism of platelets displaying defective secretion from patients with recognized functional defects.